The A3 adenosine receptor (AR) is the most recently identified subtype of receptor for the purine nucleoside adenosine, which remains poorly characterized in terms of its physiological function, particularly in the cardiovascular system. In previous studies, we have demonstrated that administering selective agonists of the A3AR effectively reduces injury in multiple different preclinical animal models of myocardial ischemia and reperfusion. One of the major advantages of A3AR therapy we have observed in our studies is that these agents are effective at doses that exert no adverse hemodynamic effects. The goal of this proposal is to further expand our knowledge of the cardioprotective actions of the A3AR by completing three highly integrated specific aims. In Specific Aim #1, we will extend our ongoing studies to explore potential mechanisms by which A3AR activation provides protection from ischemia/reperfusion injury. Building off of information gained during the previous grant cycle, we will test the hypothesis that A3AR activation attenuates lethal reperfusion injury by suppressing inflammation and neutrophil-mediated tissue injury. This hypothesis will be tested using novel genetic tools allowing for specific deletion of the A3AR in neutrophils in mice. The results of Specific Aim #2 will provide important information that will further explore the translational potential of A3AR agonists for treating ischemic heart disease. We will determine whether or not treating with A3AR agonists post-MI produces long-term preservation in cardiac performance and whether or not A3AR activation directly diminishes maladaptive remodeling responses. The final highly innovative specific aim will test the cardioprotective efficacy of new A3AR ligands that have recently been developed. We will examine the effectiveness of members of a new series of purine agonists with a modified (N)-methanocarba ring structure in place of the ribose, which we have identified as species-independent, highly selective A3AR agonists. The experiments are uniquely designed to examine whether conjugating the compound to a polymeric dendrimer will increase its cardioprotective potency and efficacy by promoting cooperative ligand-receptor interactions. Finally, we will examine the usefulness of allosteric enhancers for the A3AR. These agents act on a distinct binding site of the A3AR protein that increases the affinity of agonists acting at the orthostatic binding site for adenosine. Theoretically, allosteric enhancers offer the opportunity to target diseased tissues where the production of adenosine is increased while avoiding potential side effects caused by activation of receptors in other organs. Overall, the central objective of this proposal is to investigate the pathophysiological role of the A3AR in the cardiovascular and immune systems during myocardial ischemia/reperfusion injury. If our hypotheses are correct, we will demonstrate that A3AR activation reduces lethal reperfusion injury through anti- inflammatory mechanisms and that treating with A3AR agonists will provide, in addition to infarct size reduction, an added benefit to reduce post-infarction maladaptive remodeling. Completing this work will importantly increase our understanding of the basic biology of the A3AR and has the potential to lead to the development of novel new pharmacological strategies for treating patients with ischemic heart disease.